About two billion people are thought to be infected with the bacillus Mycobacterium tuberculosis (“Mtb”), the causative agent of tuberculosis (“TB”). The majority of those infected do not show signs of disease; however, each year about 8 million individuals develop active tuberculosis and about 2 million die (Dye et al., “Consensus Statement. Global Burden of Tuberculosis: Estimated Incidence, Prevalence, and Mortality by Country. WHO Global Surveillance and Monitoring Project,” JAMA 282(7):677-86 (1999)). Cure of tuberculosis requires months of treatment with multiple anti-infective agents. Incomplete treatment is common and encourages the emergence of multi-drug resistant (“MDR”) strains. MDR isolates are detected in all nations and prevalent in some. Infection can be acquired by sharing airspace with an individual with cavitary disease, with an infectious dose estimated at 1-10 inhaled bacilli. 
Furthermore, Mtb is a bioterrorism threat, because it has high potential for generating public fear and economic disruption. Bioterrorists could send individuals with cavitary MDR-TB through mass transit networks. Although few of the people exposed would ever develop TB, and almost none would be sickened acutely, knowledge would spread that many of those exposed would be likely to become infected, and that if MDR-TB did develop, it would be difficult to treat and lethal in up to 35% of cases among otherwise healthy individuals, even given optimal care. This would discourage congregation in subways, buses, train stations, and airports, causing economic disruption. Even a handful of cases of MDR-TB could overwhelm a regional hospital system's capacity to provide isolation. The disease rate is low, but the infection rate is high when organisms are aerosolized in shared space. Aerosolization requires no technology, only coughing.
Mtb infection can persist for decades (World Health Organization, “Tuberculosis and AIDS: Statement on AIDS and Tuberculosis,” Bull. Int. Tuberc. Lung Dis. 64:88-111 (1989); Bloom et al., “Tuberculosis: Commentary on a Re-Emergent Killer,” Science 257:55-64 (1992); Russell, “Mycobacterium Tuberculosis: Here Today, and Here Tomorrow,” Nat. Rev. Mol. Cell. Biol. 2:1-9 (2001); Raupach et al., “Immune Responses to Intracellular Bacteria,” Curr. Opin. Imm. 13:417-428 (2001)). The normal immune system creates an environment in which Mtb is not completely sterilized, yet replicates so little that 90% of immune-competent hosts who are infected with Mtb never develop overt TB. During latent infection, the primary residence of Mtb is the macrophage. The antimicrobial arsenal of the activated macrophage includes inducible NO synthase (“iNOS” or “NOS2”). At the acidic pH (<5.5) prevalent in the phagosome of activated macrophages (Schaible et al., “Cytokine Activation Leads to Acidification and Increases Maturation of Mycobacterium Avium-Containing Phagosomes in Murine Macrophages,” J. Immunol. 160(3):1290-1296 (1998)), nitrite, a major oxidation product of NO, is partially protonated to nitrous acid, which dismutates to form NO and another radical, NO2 (Stuehr et al., “Nitric Oxide. A Macrophage Product Responsible for Cytostasis and Respiratory Inhibition in Tumor Target Cells,” J.Exp. Med. 169(5):1543-1555 (1989)). Thus, mildly acidified nitrite is a physiologic antimicrobial system. Reactive nitrogen intermediates (“RNI”) may inflict not only nitrosative but also oxidative injury, as when NO combines with superoxide from bacterial metabolism to generate peroxynitrite within the bacterium (St John et al., “Peptide Methionine Sulfoxide Reductase from Escherichia coli and Mycobacterium Tuberculosis Protects Bacteria Against Oxidative Damage from Reactive Nitrogen Intermediates,” Proc. Natl. Acad. Sci. USA 98(17): 9901-9906 (2001)). Reagent NO kills Mtb with a molar potency exceeding that of most anti-tuberculosis drugs (Long et al., “Mycobacteriocidal Action of Exogenous Nitric Oxide,” Antimicrob. Agents Chemother. 43(2):403-405 (1999), Nathan et al., in In Tuberculosis, Second Edition, Rom et al., eds., Lippincott Williams & Wilkins, New York, N.Y., pp. 215-235 (2003)). In humans and mice with tuberculosis, macrophages in infected tissues and airways express enzymatically active iNOS (Facchetti et al., “Expression of Inducible Nitric Oxide Synthase in Human Granulomas and Histiocytic Reactions,” Am. J. Pathol. 154(1):145-52 (1999), Nathan, “Inducible Nitric Oxide Synthase in the Tuberculous Human Lung,” Am. J. Respir. Crit. Care Med. 166(2):13-131 (2002), Schon, Dissertation, No. 749, Linköping Universitet (2002)). Mice lacking iNOS cannot control Mtb infection (MacMicking et al., “Identification of Nitric Oxide Synthase as a Protective Locus Against Tuberculosis,” Proc. Natl Acad. Sci. USA 94(10):5243-5248 (1997)). Despite the protective effects of RNI, a small number of viable mycobacteria usually persist for the lifetime of the infected host (Hemandez-Pando et al., “Persistence of DNA from Mycobacterium Tuberculosis in Superficially Normal Lung Tissue During Latent Infection,” Lancet 356(9248):2133-2138 (2000)), and sometimes resume growth.
Persistence of Mtb in those lacking overt disease is evidenced by the emergence of TB in patients with arthritis or Crohn's disease immunosuppressed by biologicals that neutralize TNF (Keane et al., “Tuberculosis Associated with Infliximab, a Tumor Necrosis-Factor α-Neutralizing Agent,” N. Engl. M. Med. 345: 1098-1104 (2001)). More significantly, emergence of overt TB in people with subclinical Mtb infection reaches 50-80% with supervening HIV disease. Worldwide, TB may be the leading cause of death in AIDS patients (World Health Organization, “Tuberculosis and AIDS: Statement on AIDS and Tuberculosis,” Bull. Int. Tuberc. Lung Dis. 64:88-111 (1989); Bloom et al., “Tuberculosis: Commentary on a Re-Emergent Killer,” Science 257:55-64 (1992); Daley et al., “An Outbreak of Tuberculosis with Accelerated Progression Among Persons Infected with the Human Immunodeficiency Virus. An Analysis Using Restriction-Fragment-Length Polymorphisms,” N. Engl. J. Med. 326:231-235 (1992); and Lienhardt et al., “Estimation of the Impact of the Human Immunodeficiency Virus Infection on Tuberculosis: Tuberculosis Risks Re-visited? Int. J. Tuberc. Lung Dis. 1:196-204 (1997)), and TB exacerbates growth of HIV (Whalen et al., “Accelerated Course of Human Immunodeficiency Virus Infection after Tuberculosis.,” Am. J. Resp. & Crit. Care Med. 151:129-135 (1995); and Nakata et al., “Mycobacterium Tuberculosis Enhances Human Immunodeficiency Virus-1 Replication in the Lung,” Am. J. Resp. & Crit. Care Med. 155:996-1003 (1997)). Lifelong persistence of infection in immunocompetent hosts and exacerbation of infection in immunodeficient hosts suggest a dynamic balance. Inhibition of Mtb resistance pathways might tilt the balance in favor of the host, allowing the host to sterilize the pathogen and perhaps allowing conventional chemotherapy to kill the pathogen faster. Inhibition of the pathways by which Mtb resists the host might allow people who are subclinically infected to rid themselves of persistent bacilli, reduce their lifelong risk of reactivation TB, and interrupt the pandemic.
Among the most successful forms of anti-Mtb chemotherapy is that applied naturally by the host. Of these, nitric oxide (“NO”) is the only molecule known to be produced by mammalian cells that can kill tubercle bacilli in vitro with a potency (˜150 nM) comparable to that of chemotherapy. That the primary product of iNOS is mycobacteriacidal provides one type of evidence consistent with a role for iNOS in controlling tuberculosis. There are 4 more lines of evidence: (ii) immunologically activated, iNOS-expressing mouse macrophages can kill M. tuberculosis, but not if the macrophages are treated with iNOS inhibitors (Chan et al., “Killing of Virulent Mycobacterium tuberculosis by Reactive Nitrogen Intermediates Produced by Activated Murine Macrophages,” J. Exp. Med. 175:1111-22 (1992)) or bear disrupted NOS2 alleles (Ehrt et al., “Reprogramming of the Macrophage Transcriptome in Response to Interferon-γ and Mycobacterium tuberculosis: Signaling roles of Nitric Oxide Synthase-2 and Phagocyte Oxidase,” J. Exp. Med. 194:1123-1140 (2001)); (iii) iNOS is expressed in infected mouse tissues in which the growth of Mtb is restrained, but iNOS is scant when immunosuppressive drugs or genetic interventions impair host resistance (reviewed in MacMicking et al., “Identification of Nitric Oxide Synthase as a Protective Locus Against Tuberculosis,” Proc. Natl. Acad. Sci. 94:5243-5248 (1997)); (iv) healthy mice that harbor tubercle bacilli succumb abruptly to TB following ingestion of specific iNOS inhibitors (MacMicking et al., “Identification of Nitric Oxide Synthase as a Protective Locus Against Tuberculosis,” Proc. Natl. Acad. Sci. 94:5243-5248 (1997); and Chan et al., “Effects of Nitric Oxide Synthase Inhibitors on Murine Infection with Mycobacterium tuberculosis,” Infect. Immun. 63:736-40 (1995)); and (v) mice with disrupted NOS2 alleles die with fulminant TB in a few weeks, while wild type mice survive infection for ˜9 months (MacMicking et al., “Identification of Nitric Oxide Synthase as a Protective Locus Against Tuberculosis,” Proc. Natl. Acad. Sci. 94:5243-5248 (1997); Scanga et al., “The Inducible Nitric Oxide Synthase Locus Confers Protection Against Aerogenic Challenge of Both Clinical and Laboratory Strains of Mycobacterium tuberculosis in Mice,” Infect. Immun. 69:7711-7717 (2001); and Mogues et al., “The Relative Importance of T Cell Subsets in Immunity and Immunopathology of Airborne Mycobacterium tuberculosis Infection in Mice,” J. Exp. Med. 193:271-280 (2001)). When O2 is limiting, Mtb uses nitrate as an electron acceptor, generating nitrite as a byproduct (Weber et al., “Anaerobic Nitrate Reductase (narGHJI) Activity of Mycobacterium Bovis BCG In vitro and its Contribution to Virulence in Immunodeficient Mice,” Mol. Micro. 35:1017-1025 (2000)). This reaction is essential for mycobacterial proliferation in mouse lung, as judged by the failure of nitrate reductase-deficient BCG to proliferate even in immunodeficient mice (Weber et al., “Anaerobic nitrate Reductase (narGHJI) Activity of Mycobacterium Bovis BCG In vitro and its Contribution to Virulence in Immunodeficient Mice,” Mol. Micro. 35:1017-1025 (2000)). Nitrate arises from dietary sources and the action of constitutively expressed NOSs, and is thus a normal component of human blood and bronchoalveolar fluid. Nitrite regenerates NO at the mildly acidic pH pertaining in poorly oxygenated microenvironments (Stuehr et al., “Nitric Oxide: A Macrophage Product Responsible for Cytostasis and Respiratory Inhibition in Tumor target Cells,” J. Exp. Med. 169:1543-5 (1989)). Thus, Mtb needs to survive nitrosative stress generated by itself as well as by the host.
The existing armamentarium against Mtb is clinically effective when the organism is drug-sensitive and 180-270 days of drug administration are ensured by directly observed therapy. Both conditions are hard to meet. Agents are urgently needed that target additional pathways. Most approaches to antibiotic development are based on screening for compounds that inhibit the growth of the organism in pure culture, or testing inhibitors of pathways already known to be essential for growth in pure culture. Rarely has an effort been made to screen under conditions that model a critical aspect of the host-pathogen relationship. For Mtb, intraphagosomal conditions include low Fe2+, low Mg2+, and increased oxidative/nitrosative stress (Buchmeier et al., “A Parallel Intraphagosomal Survival Strategy Shared by Mycobacterium Tuberculosis and Salmonella enterica,” Molec. Microbiol. 35:1375-82 (2000); Forbes et al., “Divalent-Metal Transport by NRAMP Proteins at the Interface of Host-Pathogen Interactions,” Trends Microbiol. 9:397-403 (2001); and Nathan et al., “Reactive Oxygen and Nitrogen Intermediates in the Relationship Between Mammalian Hosts and Microbial Pathogens,” Proc. Natl. Acad. Sci. USA 97:8841-8848 (2000)). The clinical immunobiology of Mtb infection teaches that chemotherapy that is effective in vitro is less effective in the host whose immune system does not contribute to control. In the mouse, chemotherapy that works in vitro is only transiently effective in a host that lacks iNOS.
Thus, TB is the leading cause of death from a single bacterial infection and the leading opportunistic infection in HIV-infected hosts. Multiple drug resistance is rapidly spreading and exacerbates these burdens, and the threat of bioterrorism adds a new dimension to the picture. New chemotherapeutic options are needed that work faster and on additional targets than those now available. In particular, it would be useful to have more information about the genes that allow Mtb to resist host antibacterial mechanisms for the development of anti-infectives in the treatment of Mtb infection.
The present invention is directed to overcoming these and other deficiencies in the art.